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Originally published online as doi:10.1189/jlb.0804478 on November 29, 2004

Published online before print November 29, 2004
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(Journal of Leukocyte Biology. 2005;77:361-368.)
© 2005 by Society for Leukocyte Biology

Select forms of tumor cell apoptosis induce dendritic cell maturation

Sandra Demaria*,1, Fabio R. Santori*, Bruce Ng{dagger}, Leonard Liebes{dagger}, Silvia C. Formenti{ddagger} and Stanislav Vukmanovic*,§

* Departments of Pathology,
{dagger} Medicine, and
{ddagger} Radiation Oncology, and the New York University Cancer Institute, New York University School of Medicine, New York; and
§ Children’s National Medical Center, Center for Cancer and Immunology, Washington, District of Columbia

1 Correspondence: Department of Pathology, MSB-563, New York University School of Medicine, 550 First Avenue, New York, NY 10016. E-mail: demars01{at}med.nyu.edu


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ABSTRACT
 
Dendritic cells (DC) play a crucial role in initiating immune responses to tumors. DC can efficiently present antigens from apoptotic tumor cells, but apoptotic cells are thought to lack the inflammatory signals required to induce DC maturation. Here, we show that apoptosis of 67NR mouse carcinoma cells via the Fas (CD95) pathway or induced by the anticancer drug bortezomib (PS-341) but not by ultraviolet irradiation is associated with the production of maturation signals for DC. These data have important implications for the effects of chemotherapy on antitumor immunity in solid and hematologic malignancies.

Key Words: costimulation • tumor immunity • Fas/CD95 • chemotherapy


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INTRODUCTION
 
Dendritic cells (DC) can efficiently acquire and present antigens derived from apoptotic cells and stimulate antigen-specific T cell responses [1 ]. However, apoptotic cells themselves were shown not to provide the inflammatory signals required to induce DC maturation [2 3 4 ]. As apoptosis is part of normal tissue development and turnover, and antigen presentation by immature DC induces tolerance rather than activation of T cells, the uptake of apoptotic cells by DC in the absence of exogenous maturation signals is thought to be important in the maintenance of self-tolerance [5 , 6 ]. Chemotherapy drugs kill tumor cells mainly by inducing their apoptosis [7 ]. This raises the possibility that chemotherapy may actually be promoting tolerance of the patient’s own immune system to the tumor, unless maturation signals for DC are also generated. However, it is intriguing that the efficacy of DNA-expressed immunogens in vivo can be enhanced by induction of apoptosis of cells expressing them [8 9 10 ]. In addition, our prior study of breast cancer patients treated with neoadjuvant chemotherapy suggests that chemotherapy-induced tumor cell apoptosis may sometimes trigger antitumor T cell responses [11 ]. A recent study in a mouse model of cancer supports this contention [12 ]. Nevertheless, the ability of apoptotic tumor cells to induce DC maturation has not been established. Here, we used a mouse mammary carcinoma cell line, 67NR, to test whether induction of apoptosis of these cells via the Fas pathway and by treatment with the drug bortezomib (PS-341) or ultraviolet (UV) irradiation was coupled with the production of maturation signals for DC. Our results show, for the first time, that cancer cell death induced by some but not all apoptosis stimuli can result in DC maturation.


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MATERIALS AND METHODS
 
Construction of Fas-estrogen receptor (ERT) hybrid protein
The cDNA encoding for the death domain and transmembrane region of murine Fas (provided by Akira Kakizuka, Kyoto University, Japan) was fused to the cDNA encoding for the ligand-binding domain (LBD) of a G525R mutant murine ERT, which binds selectively to 4-hydroxytamoxifen (4HT) [13 ] (provided by Gerard I. Evan, University of California San Francisco, Cancer Center; see Fig. 1A ). The hybrid protein gene was subcloned under the control of the cytomegalovirus promoter into the cloning site of the retroviral vector PALLINO (provided by Giorgio Inghirami, New York University School of Medicine, New York), which also expresses enhanced green fluorescent protein (EGFP) [14 ]. PALLINO/Fas-ERT was transfected into Phoenix Eco (American Type Culture Collection, Manassas, VA, Inventory No. SD 3444) cells and supernatants containing the recombinant virus used to infect 67NR cells.



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  		Figure 1. Induction of apoptosis of 67NR/Fas-ERT (G4) cells via the Fas pathway by treatment with 4HT. (A) Construction of Fas-ERT chimeric proteins. The mutant LBD of the mouse ERT [amino acids (aa) 287–599] is fused at the C terminus of mouse Fas (aa 135–305), which includes the transmembrane region (shaded box) and death domain (aa 201–286). (B) Differential sensitivity of G4 cells to 4HT and 17-ß-estradiol (E2). G4 and parental 67NR cells were cultured for 18 h in triplicate wells in the presence of 4HT or E2 at the indicated concentrations. Cell viability was assessed using the MTT-cleavage assay. (C) Activation of caspase-3. G4 cells were cultured in the presence of 4HT (0.1 µM) for indicated times. Cleavage of caspase-3 substrate DEVD-pNA was measured as increase in optical density absorbance at 405 nm [OD (A405)]. (D) G4 cells were incubated with 4HT (1 µM) followed by TUNEL assay and flow cytometric analysis. DNA fragmentation was detected in 48% and 87% of the cells by 4 h and 8 h, respectively. Data are representative of two experiments.

Cells and cell culture
67NR is a BALB/c mouse-derived mammary carcinoma cell line (provided by Fred Miller, Michigan Cancer Center, Ann Arbor). 67NR cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen Corp., Carlsbad, CA) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 x 10–5 2-mercaptoethanol, and 10% fetal bovine serum (FBS; Gemini Bio-Products Woodland, CA). These cells were found to be free of contamination by Mycoplasma and by 12 common viruses (essential virus profile, Charles River Laboratories, Wilmington, MA). 67NR cells are Fas (CD95)-negative, as confirmed by immunostaining with anti-CD95 monoclonal antibody (mAb; BD PharMingen, San Diego, CA). Following infection with the Fas-ERT encoding retrovirus, strongly EGFP-positive 67NR/Fas-ERT cells were sorted and subcloned to obtain cells with stable expression of the chimeric protein. The subclone G4 was selected for further experiments. Immature DC were obtained by culturing bone marrow cells derived from BALB/c wild-type or recombination activation gene-knockout (RAG-KO) mice with granulocyte macrophage-colony stimulating factor for 7–8 days as described [15 ].

Preparation of apoptotic and necrotic tumor cells
Apoptotic G4 cells were prepared by incubation with 4HT (0.1 µM) for 5–6 h to induce apoptosis via the Fas pathway (G4-Fas). G4 cells were also irradiated using a UVB lamp (60 mJ at 2 mJ/cm2/s), followed by culture at 37°C for 20 h as described [16 ], at which time caspase-3 activation was maximal (G4-UV). Apoptotic 67NR cells were prepared by incubation with PS-341 (provided by Millenium Pharmaceuticals, Cambridge, MA; 0.1 µM) for 48 h (67NR-PS341). Cell viability was assessed using the 3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-cleavage assay [17 ]. Caspase-3 activation was detected using the CPP32/caspase-3 colorimetric protease assay kit according to manufacturer’s instructions (MBL International, Watertown, MA). Briefly, 67NR cells were lysed, and equal amounts of proteins were incubated with the caspase-3 substrate Asp-Glu-Val-Asp (DEVD)-p-nitroanilide (pNA). Cleavage of DEVD-pNA was measured as increase in absorbance at 405 nm. Occurrence of DNA strand-breaks was assessed using the deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay using tetramethyl-rhodamine-dUTP (Roche Molecular Biochemicals, Indianapolis, IN) followed by flow cytometry analysis. Alternatively, enrichment of nucleosomes in the cytoplasm of apoptotic cells was measured using the cell death detection enzyme-linked immunosorbent assay (ELISA; Roche Molecular Biochemicals). To evaluate nuclear morphology, cells were transferred onto microscope slides using Cytospin 2 (Shandon, Thermo Electron Corp., Waltham, MA), fixed with Carnoy’s fixative (75% ethanol, 25% acetic acid), and stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO), followed by examination using a Nikon Eclipse TE-300 microscope equipped with Spot v3.59 software (Diagnostic Instruments, Sterling Heights, MI). Necrotic cells were prepared by four cycles of rapid freezing and thawing as described [2 ].

Inhibition of apoptosis
Pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (zVAD-FMK), caspase-8 inhibitor Ile-Glu-Thr-Asp (IETD)-FMK, and caspase-9 inhibitor Leu-Glu-His-Asp (LEHD)-FMK (Calbiochem, San Diego, CA) were used at 40 µM. 67NR and G4 cells were incubated at 104/well in a 96-well flat-bottom plate in triplicate wells. Caspase inhibitors were added 1 h before exposure to 4HT (0.1 µM), PS-341 (0.1 µM), or UV irradiation. Cell death was measured by the cytotoxicity detection kit (Roche Molecular Biochemicals), according to the manufacturer’s instructions. Percentage of apoptosis was calculated as: (AbsD±CI–AbsCI/AbsTOT–Absmed) x 100, where AbsD+CI is the absorbance (Abs) measured at 490 nm from wells incubated in the presence of drug and caspase inhibitor, AbsCI is the Abs from wells incubated with the caspase inhibitor alone, AbsD is the Abs from wells incubated with the drug alone, AbsTOT is the Abs from wells lysed with 1% Triton X-100, and Absmed is the Abs from wells incubated with medium alone. Similar results were obtained using the MTT-cleavage assay to estimate cell death.

Maturation of DC
DC were cultured with live, apoptotic, or necrotic tumor cells at a ratio of 1:1–1:2 DC/tumor cells or with lipopolysaccharide (LPS; 20 ng/ml) as control for 20 h. Apoptotic G4 and 67NR cells were prepared by UV irradiation and culture for 20 h or by culture with 4HT for 6 h and with PS-341 for 48 h as described above. Apoptotic cells were harvested by gentle pipetting and washed three times by centrifugation in culture medium before coculture with DC to remove any residual drug. DC were then harvested in cold phosphate-buffered saline containing 1% FBS and 5 mM EDTA, washed, and analyzed by triple staining with mAb against mouse CD45-Cy-Chrome, CD11c-fluorescein isothiocyanate (FITC), and CD40-phycoerythrin (PE), CD80-PE, CD86-PE, and Iad-PE (BD PharMingen). Cells were analyzed using a FACScan flow cytometer. Differences in the expression of CD40, costimulatory, and major histocompatibility complex (MHC) molecules between DC cultured with medium alone and with tumor cells were tested using a one-tailed Student’s t-test with a significance level of P < 0.05.

Mice vaccination
Five- to 6-week-old wild-type and RAG-KO female BALB/c mice were obtained from Taconic Animal Laboratory (Germantown, NY). All experiments were approved by the Institutional Animal Care and Use Committee of New York University. 67NR cells were induced to apoptosis by UV irradiation or incubation with PS-341 as described above. BALB/c female mice were vaccinated subcutaneously (s.c.) with DC cocultured or not with apoptotic 67NR cells at a ratio of 1:1 for 18–20 h. After 8 or 9 days, mice were challenged with a tumorigenic inoculum of 67NR cells (5x104) s.c. in the opposite flank and followed for tumor development.

Phagocytosis assay
The phagocytosis assay was performed as described previously [2 ]. Briefly, 67NR cells were labeled red with PKH26 following the manufacturer’s instructions (Sigma-Aldrich). Labeled 67NR cells were then induced to apoptosis by UV irradiation or by culture in the presence of PS-341 for 48 h as described above. After washing, apoptotic cells were cocultured with DC at a ratio of 1:1 for 3 h, at 4°C or 37°C, followed by staining with CD11c-FITC for 30 min at 4°C. Phagocytosis of apoptotic cells by DC was defined by the percentage of double-positive cells by fluorescein-activated cell sorter (FACS) analysis.


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RESULTS
 
Induction of apoptosis of 67NR carcinoma cells expressing a chimeric Fas-ERT receptor by treatment with 4HT
To test whether it is possible to induce an apoptosis of tumor cells coupled to the production of maturation signals for DC, we used as a model the mouse mammary carcinoma 67NR. As in an in vivo system, Fas-induced apoptosis of cells expressing a vaccine antigen has been shown to markedly enhance specific T cell responses [9 ], a derivative of 67NR cells (G4) was generated in which Fas-mediated apoptosis can be induced via a hybrid protein consisting of the death domain of Fas fused to the LBD of a mutated ERT, Fas-ERT (Fig. 1A ). Apoptosis of G4 cells was rapidly induced by incubation with 10 nM or higher 4HT, which results in cross-linking of Fas death domains, whereas 17-ß-estradiol (E2) was ineffective (Fig. 1B) . This allowed selective induction of the tumor cell apoptosis that was regulated by the addition of 4HT, as the cells were not sensitive to the estrogens present in the serum used for cell culture. Activation of caspase-3 and occurrence of DNA fragmentation further confirmed that G4 cells were dying by apoptosis in response to 4HT (Fig. 1C and 1D) .

Induction of apoptosis of 67NR carcinoma cells by treatment with PS-341
Apoptosis of 67NR cells induced by the drug PS-341 was also studied. PS-341 is a novel, reversible inhibitor of the 26S proteasome, which leads to arrest into the G2-M phase of the cell cycle followed by apoptosis of proliferating but not quiescent cells [18 ]. PS-341 does not exhibit significant bone marrow toxicity, is active against several types of cancer, has been approved recently for the treatment of multiple myeloma, and is under evaluation in clinical trials for solid and hematologic tumors [19 , 20 ]. 67NR cells were sensitive to PS-341 at concentrations above 1 nM, and the majority of cells undergoes apoptosis within 48 h at 100 nM concentration of the drug (Fig. 2A ). At this concentration of PS-341, the caspase-3 activation peaked at approximately 30 h (Fig. 2B) . DNA fragmentation was detectable within 24 h at PS-341 concentrations of 10 nM and above (Fig. 2C) .



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  		Figure 2. Induction of apoptosis of 67NR cells by treatment with PS-341. (A) Sensitivity of 67NR cells to PS-341 cytotoxicity. 67NR cells were cultured for 24 h or 48 h in triplicate wells in the presence of PS-341 at the indicated concentrations. Cell viability was assessed using the MTT-cleavage assay. (B) Activation of caspase-3. 67NR cells were cultured in the presence of PS-341 (0.1 µM) for indicated times. Cleavage of caspase-3 substrate DEVD-pNA was measured as increase in absorbance at 405 nm [OD (A405)]. (C) 67NR cells were incubated with PS-341 at the indicated concentrations for 24 h followed by lysis of the cell pellet and determination of the nucleosomes in the cytoplasmic fraction by ELISA.

Activation of different death pathways in 67NR cells by PS-341, 4HT, and UV treatment
Different kinetics of Fas- and PS-341-induced apoptosis in 67NR cells suggested that they may involve different biochemical pathways. Although the molecular mechanisms of activity of PS-341 are still under study, a recent report shows that PS-341 triggers a dual apoptotic pathway in myeloma cells involving the mitochondrial pathway as well as a Fas/caspase-8-dependent pathway [21 ]. To test whether apoptosis induced by PS-341 involves the activation of the Fas/caspase-8 pathway in our experimental model, we first compared the sensitivity of 67NR and G4 cells to this drug. Consistent with the hypothesis that PS-341 can lead to activation of the Fas-mediated pathway, death of G4 cells in response to PS-341 was markedly faster than death of parental Fas-negative 67NR cells (Fig. 3A ). The increased sensitivity of G4 cells was specific to PS-341, as no increase in sensitivity to docetaxel, paclitaxel, topotecan, and cisplatin was seen (S. Demaria, B. Ng, L. Liebes, Peter Elliott, and S. C. Formenti, Enhanced sensitivity of mammary carcinoma cell lines to the effects of the proteasome inhibitor PS-341 when Fas signal transduction is perturbed. 24th Annual San Antonio Breast Cancer Symposium, 2001, Abstract 549). Next, the relative contribution of the Fas/caspase-8 and mitochondrial/caspase-9 pathways to apoptosis of 67NR and G4 cells was investigated by testing the sensitivity of PS-341-induced apoptosis to specific caspase inhibitors. The caspase-8 inhibitor IETD-FMK almost completely inhibited the PS-341-induced death of G4 and 67NR cells (Fig. 3B and 3C) . In contrast, minimal inhibition was seen in the presence of the caspase-9 inhibitor LEHD-FMK. Therefore, the Fas/caspase-8-mediated pathway is crucial for PS-341 effect in these cells.



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  		Figure 3. Role of the Fas/caspase-8 and mitochondrial/caspase-9 apoptosis pathways in the death of 67NR cells induced by PS-341 and 4HT and UV irradiation. (A) Death of G4 cells treated with PS-341 is faster in comparison with 67NR cells. 67NR ({square}) and G4 (•) cells were cultured for 24 h in triplicate wells in the presence of PS-341 at the indicated concentrations. Cell viability was assessed using the MTT-cleavage assay. Although 67NR and G4 cells die in response to doses of PS-341 >10 nM, the death of G4 cells is faster, as it is virtually complete within 24 h compared with 67NR cells, which require 48 h (see Fig. 2 ). (B–E) Inhibition of apoptosis of G4 cells (B and D) and 67NR cells (C and E) induced by incubation with PS-341 (B and C), 4HT (D), or UV irradiation (E) by the pan-caspase inhibitor zVAD-FMK, the caspase-8 inhibitor IETD-FMK, and caspase-9 inhibitor LEHD-FMK. Total cell death in the absence (NONE) or presence of caspase inhibitors was measured 6 h after addition of 4HT or 24 h after addition of PS-341 or UV irradiation. Results are representative of two to four experiments. (F and G) Nuclear morphology of 67NR cells stained with Hoechst 33342. Chromatin condensation is evident 24 h following UV irradiation (F) but not mock treatment (G). (H) Activation of caspase-3 following UV treatment of 67NR cells and culture for indicated times. Cleavage of caspase-3 substrate DEVD-pNA was measured as increase in absorbance at 405 nm [OD (A405)].

Apoptosis induced in G4 cells by cross-linking of Fas death domain mediated by 4HT was partially and equally inhibited by IETD-FMK and LEHD-FMK (Fig. 3D) , indicating that G4 behave like type II cells and do not generate sufficient active caspase-8 at the death-inducing signaling complex to use a mitochondria-independent apoptosis pathway [22 ].

As tumor cells induced to apoptosis by UV treatment have been shown to be unable to induce DC maturation [2 ], the effects of UV irradiation on 67NR and G4 cells were also tested. It is interesting that death of UV-treated 67NR and G4 cells was only minimally inhibited by caspase inhibitors, including zVAD-FMK, despite the fact that the cells showed clear, morphologic features of apoptosis and caspase-3 activation (Fig. 3E 3F 3G 3H , and data not shown). These findings are suggestive of the involvement of a major caspase-independent pathway in the UV-induced apoptosis of these tumor cells. A possible candidate may be the mitochondrial apoptosis pathway mediated by apoptosis-inducing factor (AIF) but independent of other caspases [23 , 24 ]. Overall, these results indicate that PS-341, 4HT, and UV treatment activate different death pathways in 67NR cells.

Induction of DC maturation by exposure to apoptotic 67NR carcinoma cells
Having established the characteristics and different degrees of caspase dependence of Fas-, PS-341-, and UV irradiation-induced death of 67NR and G4 cells, we tested the ability of apoptotic cells to induce DC maturation. Mature DC up-regulate MHC, CD40, and costimulatory molecules, which are necessary for activation of T cells; therefore, live and dead 67NR cells were cocultured with bone marrow-derived, immature DC, as described previously [3 , 4 ]. Staining of DC to determine the induction of maturation markers was then performed. To minimize DC manipulations that could activate them [3 ], DC were not purified from tumor cells after coculture but stained directly with a cocktail of mAb and samples gated on CD11c+CD45+ DC (Fig. 4A ). Exposure of DC to live tumor cells did not induce the expression of CD40 or the up-regulation of CD86 and MHC class II molecules Iad (Fig. 4C 4D 4E 4F) . Although there was a small increase in the expression of CD80, this was not significant. Necrotic tumor cells, as previously reported [2 3 4 ], were good inducers of DC maturation. Induction of tumor cell apoptosis by UV irradiation of G4 (Fig. 4C 4D 4E 4F) or 67NR cells (data not shown) did not lead to significant up-regulation of any of the DC maturation markers analyzed, as reported by Sauter et al. [2 ]. In contrast, exposure of DC to tumor cells induced to apoptosis via the Fas pathway and by PS-341 resulted in up-regulation of CD40 and costimulatory molecules that were significant and comparable with the effect of necrotic tumor cells (Fig. 4B 4C 4D 4E 4F) . LPS, as expected, was the most powerful inducer of DC maturation. Apoptotic cells triggered via the Fas pathway were at least as effective as LPS and superior to necrotic cells in inducing the up-regulation of Iad molecules, whereas apoptotic cells triggered by PS-341 did not induce a significant Iad up-regulation (Fig. 4B and 4F) . Overall, these data indicate that different inducers of apoptosis affect the capacity of apoptotic tumor cells to induce DC maturation.



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  		Figure 4. Apoptosis of 67NR cancer cells induced via the Fas pathway or by the anticancer drug PS-341 but not by UV irradiation is coupled to the production of maturation signals for DC. (A) Gating of triple-stained DC. To analyze the expression of maturation markers on DC following coculture with live or dead carcinoma cells, samples were gated on CD11c+ and CD45+ cells to exclude CD11c–CD45+ myeloid precursors (15–25%) present in the DC preparations (left panel) and CD11c–CD45– 67NR cells (right panel). Apoptotic G4 cells still express EGFP positivity detectable in the FL1 channel, but they can be separated easily from DC, as they are CD45– (middle panel). (B) Expression of CD40, CD80, CD86, and Iad on CD11c+CD45+ DC cultured in medium alone (thin line) versus DC cultured with G4 cells induced to apoptosis via the Fas pathway (bold line). Cells stained with isotype-control antibody (shaded histograms). Percentages of cells included in the gate are shown. (C) Induction of CD40 expression on DC cocultured with carcinoma cells. DC were cultured for 20 h in medium alone (–) or in the presence of live or necrotic (NECRO) G4 and 67NR cells, G4 cells induced to apoptosis via the Fas pathway (G4-Fas) or by UV irradiation (G4-UV), or 67NR cells induced to apoptosis by PS-341 (67NR-PS341). Numbers indicate the percentage of positive cells and are expressed as the mean ± SD of three experiments. In the presence of LPS, 90–95% of DC acquired the expression of CD40. The percentage of DC expressing CD40 following incubation with cancer cells was much smaller, but only dead and not live cancer cells were able to induce CD40. (D–F) Up-regulation of CD80, CD86, and Iad on DC cocultured with carcinoma cells. As low levels of these molecules are expressed on immature DC, and upon maturation, there is an increase in the mean fluorescence intensity (MFI) of low and high expressor subsets as well as in the percentage of cells with high expression, the MFI of the entire population is shown. Data are expressed as the mean ± SD of three experiments. (C–F) *, Statistically significant differences (P<0.05) between expression on DC exposed to tumor cells and control DC incubated in medium alone. Culture of DC in the presence of 4HT (100 nM) and PS-341 (10 nM) did not induce an increase in expression of maturation markers by DC, excluding the possibility that the maturation observed is a result of drug carry-over.

It has been suggested previously that a high ratio (5:1) of apoptotic cells to DC is required to induce DC maturation [16 ]. However, when the ratio of apoptotic tumor cells to DC was increased from 1 to 5, the expression of maturation markers by DC did not increase further but actually slightly declined (data not shown). We found optimal induction of DC maturation at a ratio of 1:1–2:1 apoptotic tumor cells to DC. Recovery of viable DC in these cultures was good (~90%) and comparable with the control, ruling out the possibility that the maturation signals were generated by the dying DC themselves. Taking advantage of the selective induction of Fas-mediated apoptosis in G4 cells by 4HT, the impact of the timing of apoptosis on DC maturation was studied. 4HT was added to live G4 cells at the beginning of the coculture with DC, allowing the apoptotic process to take place during the exposure to DC. There was no significant difference in induction of DC maturation in these cultures compared with cultures where apoptotic G4 cells were added to DC after 6 h of exposure to 4HT and washing (data not shown). 4HT alone did not have any effect on DC maturation or viability.

Vaccination of mice with DC exposed to 67NR cells induced to apoptosis by PS-341 but not by UV can protect mice from tumor challenge
Next, we tested whether DC exposed to apoptotic tumor cells can indeed mature into effective antigen-presenting cells capable of triggering antitumor T cell responses in vivo. To this end, mice were vaccinated with DC exposed to apoptotic 67NR cells and then challenged with a tumorigenic inoculum of viable 67NR cells. As EGFP is expressed by G4 cells and has been shown to be immunogenic in BALB/c mice [25 ], to exclude its effect in inducing an antitumor immune response, experiments were performed with a vaccine consisting of DC exposed to 67NR cells dying by PS-341-induced apoptosis. Mice vaccinated with DC cocultured with apoptotic 67NR cells were protected from tumor development (Fig. 5 ). At least 105 DC were needed to elicit protective antitumor immunity. Induction of DC maturation by the apoptotic tumor cells was crucial for the triggering protective antitumor immunity, as vaccination with DC exposed to UV-treated apoptotic 67NR cells did not protect mice from tumor development (Fig. 5C) . Uptake of UV- and PS-341-treated 67NR cells by DC at 37°C was comparable with a 3-h or longer phagocytosis assay (data not shown). Uptake was markedly reduced if the incubation of DC with apoptotic tumor cells was performed at 4°C, indicating that dying tumor cells were not bound nonspecifically. Therefore, the differential ability of DC exposed to 67NR cells undergoing UV- and PS-341-induced apoptosis to vaccinate mice is not a result of incubation conditions favoring uptake of PS-341-treated 67NR cells. It is important that vaccination did not protect RAG-deficient mice from tumor development, indicating that an intact immune system was required for this effect (data not shown). When the vaccinated, tumor-free mice from the first experiment (Fig. 5A) were rechallenged with 67NR cells 82 days after the first challenge, they did not develop tumors, whereas all of the control mice had palpable tumors by day 7 (data not shown). These findings suggest that vaccination with DC/67NR-PS-341 can elicit a long-lasting, antitumor-immune response.



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  		Figure 5. Vaccination with DC exposed to 67NR cells dying of PS-341- but not UV-induced apoptosis protects mice from tumor development. (A) DC incubated with medium alone or with apoptotic 67NR cells (DC/67NR-PS-341) were injected s.c. into syngeneic BALB/c mice (106 cells/mouse; five mice/group). After 8 days, mice were challenged with live 67NR cells. Tumors were growing in 80% of control-untreated ({blacktriangleup}) and DC-vaccinated ({circ}) mice by days 11 and 13 postchallenge, respectively. In contrast, mice vaccinated with DC pulsed with apoptotic 67NR cells ({diamondsuit}) remained tumor-free. (B) Protection from tumor development depends on the dose of DC/67NR-PS-341 vaccine. Mice (five mice/group) were vaccinated with DC incubated with medium alone ({circ}; 5x105 cells) or with apoptotic 67NR cells (DC/67NR-PS-341), then challenged with live 67NR cells 8 days later. Vaccination with 5 x 105 ({diamondsuit}) and 1 x 105 ({blacktriangleup}) DC/67NR-PS-341 was effective at protecting 80% and 40% of the mice, respectively, whereas 2 x 104 DC/67NR-PS-341 ({blacksquare}) did not have any effect. (C) Mice (five mice/group) were vaccinated with 5 x 105 DC incubated with medium alone ({circ}), with PS-341-treated apoptotic 67NR cells ({diamondsuit}), or with UV-treated apoptotic 67NR cells ({blacktriangleup}) and were then challenged with live 67NR cells 8 days later. Only vaccination with DC exposed to tumor cells induced to apoptosis by PS-341 but not by UV can protect mice from tumor development.


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DISCUSSION
 
The data presented here show that apoptosis can have distinct, immunological significance, depending on how it is triggered. PS-341 and Fas-mediated apoptosis of 67NR cancer cells correspond to the "inflammatory" apoptosis defined by Restifo [26] who proposed the existence of different types of apoptosis depending on how it is initiated and in what cell type it occurs. According to his model, apoptosis occurring during development or tissue turnover is immunologically "bland," whereas apoptosis following viral infections or Fas ligation can be intrinsically coupled to the production of inflammatory signals and trigger powerful immune responses [26 ].

Other investigators have emphasized the qualitative difference between apoptosis and necrosis in providing activation signals to the immune system [2 3 4 ]. In these studies, apoptotic cells were found to be unable to induce DC maturation, but a single inducer of apoptosis was studied. Our data are in agreement with Sauter et al. [2 ] that UV-induced apoptosis is not coupled to the production of maturation signals for DC. Although it has been reported by Rovere et al. [16 ] that cells undergoing UV-induced apoptosis, when in high numbers, can induce DC maturation, we did not detect DC maturation even when increasing the number of UV-treated 67NR cells cultured with DC (data not shown). It is possible that UV can trigger different events in different cell types (e.g., lymphoma vs. carcinoma). Alternatively, the source of DC used may explain the difference in results, as Rovere et al. [16 ] used a long-term DC line rather than DC differentiated from bone marrow in short-term cultures. The inability of UV-induced apoptosis to trigger DC maturation is consistent with the known inhibition of cell-mediated immune responses and induction of tolerance in contact hypersensitivity models by UV irradiation (reviewed in ref. [27 ]).

Although we cannot exclude the occurrence of postapoptotic (secondary) necrosis of tumor cells during the culture with DC, it is clear that following UV-induced apoptosis, no soluble factors that induce DC maturation are released by postapoptotic cells, as previously reported by others [2 ]. In contrast, even when Fas-mediated apoptosis of G4 cells is induced during the culture with DC, the latter are induced to mature. Therefore, it is unlikely that the degree of membrane integrity is a major factor in determining the immunogenicity of apoptotic cells. Apoptosis is an active process regulated by caspases and mitochondrial membrane permeabilization during which some factors may be degraded, whereas others can be up-regulated or activated (reviewed in ref. [28 ]). It is interesting that up-regulation of heat-shock proteins on stressed apoptotic leukemia cells can increase their ability to activate DC [29 ]. Identification of the maturation signals produced/released by dying tumor cells is crucial for understanding the immunological effects of different pathways of apoptosis. The two main pathways of apoptosis are triggered through surface death receptors (extrinsic) and the mitochondria (intrinsic). The release of AIF from mitochondria can induce DNA fragmentation and apoptosis independently of effector caspases [30 , 31 ]. Caspase-2 has been identified recently as the initiator caspase in AIF-mediated apoptosis induced in mammary carcinoma cells by the nuclear receptor-interacting factor 3 (NRIF3) family of transcriptional coregulators [24 ]. As caspase-2 is relatively resistant to inhibition by zVAD-FMK [24 ], the lack of inhibition of UV-induced apoptosis by this pan-caspase inhibitor in 67NR cells (Fig. 3E) does not exclude a role for caspase-2 in UV-induced death of these cells. Consistent with this possibility, caspase-2 has been shown to be required for the UV-induced death of some cells [32 ]. Although apoptosis mediated by AIF can proceed in the absence of effector caspases, release of cytochrome c from mitochondria and activation of caspase-9 and downstream effector caspases can amplify the apoptotic process in this pathway, similarly to the amplification of receptor-mediated apoptosis [23 , 33 ]. This can explain why we detected activation of caspase-3 following UV treatment of 67NR cells despite the inability to block cell death with caspase inhibitors (Fig. 3H) . We are currently investigating whether the production of maturation signals for DC is restricted to apoptosis mediated via caspase-8-dependent pathway(s) or can also be induced during apoptosis initiated by caspase-2 in 67NR and other tumor cells.

The induction of an inflammatory apoptosis in cancer cells by an antineoplastic drug such as PS-341 and via the Fas pathway, which contributes to the effects of other chemotherapy agents [7 ], may have important therapeutic implications. Apoptosis is a common event in many growing tumors and may contribute to the induction of immune tolerance by mimicking the bland apoptosis of normal tissue turnover [34 ]. In support of this hypothesis, cross-presentation of tumor-derived antigens by DC was shown to be a dominant mechanism of tolerance induction during lymphoma progression as well as in solid malignancies [35 , 36 ]. It is intriguing to consider whether chemotherapy drugs such as PS-341, which are capable of inducing an inflammatory apoptosis of tumor cells, could be used to enhance tumor immunogenicity.


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ACKNOWLEDGEMENTS
 
This work was supported by Grant K08 CA89336 from National Institutes of Health (NIH)/National Cancer Institue, by a grant from the Speaker’s Fund for Biomedical Research: Toward the Science of Patient Care, awarded by the City of New York; by a Breast Cancer Research Award from The Chemotherapy Foundation made possible by The Joyce and Irving Goldman Family Foundation (to S. D.); and by Grant NIH AI41573 and an award from the Irving Weinstein Foundation (to S. V.). We thank Anne Marie Yang for technical assistance, John Hirst for FACS analysis, Nina Bhardwaj for reading the manuscript, and Herbert Samuels for helpful discussion of the apoptosis pathways.

Received August 27, 2004; revised October 15, 2004; accepted November 7, 2004.


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